Homeostasis of multicellular organisms is controlled not only by proliferation and differentiation of cells but also by cell death. Physiological cell death mostly proceeds by apoptosis, a process which includes blebbing of the plasm membrane, condensation and segmentation of the nuclei and cytoplasm, extensive fragmentation of chromosomal DNA into nucleosome units, and cellular fragmentation into membrane apoptotic bodies which minimize the leakage of cellular constituents from the dying cell. Although apoptosis is necessary for normal development, pathological cell death occurs when the apoptotic process is either impaired or overactive contributing to a number of disease conditions, such as hepatitis-C, autoimmune disorders, diabetes, acute pancreatitis and numerous other disorders. Normalizing or modulating the apoptotic process; ie allowing apoptosis to carry out its important biological processes such as morphogenesis, tissue homeostasis, elimination of damaged or virally infected cells and the elimination of self-reactive clones from the immune system, would lead to therapeutic treatment for many of these degenerative diseases and disorders.
Apoptosis proceeds by two known triggering mechanisms; the Fas-antigen/ligand, and Tumor necrosis factor alpha(TNF-.alpha.)-Tumor necrosis factor receptor-1 (TNFR1) killing pathways. Molecular and cellular characterization of Fas antigen (CD95), a cell-surface protein recognized by cytotoxic monoclonal antibodies, has led to its identity as a receptor for Fas ligand (FasL). FasL binds to Fas, which results in triggering target cell apoptosis. Exaggeration of this system leads to pathological cell death and tissue destruction. Upregulating this antigen relates to many pathological situations in non-lymphoid organs, the Fas signaling pathways remain elusive. It has been shown that Fas-induced apoptosis of primary cultured hepatocytes requires an inhibitor of translation or protein kinase inhibitors, suggesting that two distinct pathways of Fas signaling exist in hepatocytes.(Rouquet N., et al., Biochem. Biophys. Res. Com. 229:27-35 (1996)).
In the liver, apoptosis is a physiological process involved in the clearance of injured cells and hemostatic control. However, in patients with viral fulminant hepatitis, or with nonacute liver disease, dramatic liver failure or secondary cirrhosis results from the death of hepatocytes, which is caused by the activation of both 55-kD tumor necrosis factor receptor (TNFR1) or CD95 (Fas/Apo-1). These receptors are independent and differentially regulated triggers of murine apoptotic liver failure (Leist M.,et al., Mol. Med. 2:109-124(1996)).
In chronic and filminant hepatitis-C, Fas expression is upregulated in the hepatocytes, especially near liver infiltrating lymphocytes. (Okazaki M., et al. Dig. Dis. Sci. 41:2453-2458 (1996)). The same correlation has been established with hepatitis-B virus infection. (Mochizuki K, et al. Hepatol. 24:1-7(1996)). (Ando K., et al., J. Immunol. 158:5283-529(1997)) studied the killing mechanism of bystander cells, zo which have upregulated Fas antigen. They found that killing of the antigen presenting sensitive cells is mediated by Fas ligand. Okazaki et al., suggests that hepatitis Fas expression is associated with persistent infection of hepatitis-C virus. Fas expression is upregulated in and the same correlation has been established by Mochizuki et al., for hepatitis-B virus infection.
It has also been shown that effective bystander killing requires intracellular contact between CTLs and target cells. TNF-.alpha. released from the CTLs mediates lysis of the bystander cells without a dose cell-cell contact. (Ando K., et al) Hepatic Fas expression was reduced significantly after treatment compared with the pretreatment values. Thus, a potential pathogenic treatment would require an antiapoptotic effect.
To date treatment of fulminant hepatitis relies mainly on orthotopic liver transplantation, which is limited by immunological complications and graft availability. Moreover, Fas mediated apoptosis occurs in hepatic allografts and is correlated with acute rejection. (Rouquet N., et al., Curr. Bio., 6:1192-1195(1996)). Thus, the task of a pathogenic treatment which unravels the Fas antigen blocking mechanism would allow for the design of an efficient treatment for apoptosis associated with viral hepatitis-C, acute liver failure and allograft rejection.
The development of biological response modifiers to increase macrophages mediated cytotoxicity by means of inducing TNF-.alpha. production and nitric oxide (NO) expression has become an area of great interest. In this respect, muramyl peptides are regarded as a most promising stimulaters. D-peptidoglycans namely N-acetyl-D-glucosaminyl-(.beta.1-4)-N-acetylmuramyl-L-alanyl-D-isoglutamin e (GMDP) have been proposed as the cytotoxic agents capable of eliminating cancer cells and/or virus infected cells. (Ovchinnikov, et al. U.S. Pat. No. 4,395,399).
Later, a considerable amount of research was done in order to increase the immunostimulatory efficacy of muramyl peptides (GMDP) by creating new compositions. The new compositions included the addition of lipopolysaccharide (LPS), polysaccharide such as polylactide, in combination with GMDP and MDP to increase macrophage mediated cytotoxidty (Seyler et al., lnt. J. Immunopharmac., Vol.18, N6.pp385-392, (1996)). Zink-proline salt was proposed to improve homogenecity of the artificial lipid impurities in GMDP. (Grubhofer, U.S. Pat. No. 5,773,011). Additional, peptide has been affixed to muramyl peptide in order to increase their immunogenicity (Le Frander et al., U.S. Pat. No. 4,401,659). Neoglucoproteins having affinity to macrophages or monocytes has also been proposed as an immunogenic component bound to muramyl peptides. (Monsigny et al., U.S. Pat. No. 4,801,578). Contrary to prior art, the present invention strives to decrease or eliminate LPS and polysacchrides. The inventors have found that decreasing levels of these components increases the newly discovered apoptosis regulating properties of GMDP. Thus the inventors have demonstrated that the purity level of the GMDP effects its ability to protect cells and modulate apoptosis. Regulating negative apoptosis, which occurs in many chronic viral infections and neurodegenerative conditions, requires an absolutely opposite task that the prior art has considered for GMDP, that is the inhibition of the cellular immunotoxicity (FAS antigen mediated by CD8 lymphocytes and macrophages) and humoral cytotoxicity (TNF-.alpha. mediated). For example, negative apoptosis is a main pathogenic pathway leading to liver necrosis and cirrhosis. Under such conditions, additional immunostimulation exerts detrimental effects with dangerous exaggeration of liver necrosis. Activated macrophages have been shown to be cytotoxic not only for isolated liver cells, but also in eight patients with hepatitis (Mizugoshi et al., Hepatogastroenterology, 1981;28:250-253). Moreover, the only treatment for hepatitis-C (based on the conventional strategy of macrophages stimulation by Interferons) failed to provide satisfactory results in these patients. Moreover, a high percentage of these patients experienced detrimental effects of this immunostimulation with prominent toxicity. Clinical symptoms of increased fatigue, viral load, and acceleration of liver necrosis is noticed as a consequence of the powerful immunostimulator, interferons. In direct opposition to conventional hepatitis-C treatments and prior use of GMDP, the present invention is not an immune stimulator but accomplishes the task of concurrent inhibition of both TNF-.alpha. and Fas antigen mediated cytotoxicity without extensive detrimental immunostimulation.
Until now, few compositions have been proposed to regulate aberrant apoptosis. Nakai et al. developed a method of the treatment liver cirrhosis based on carbostyril derivative and acted as apoptosis regulator in the patients with liver cirrhosis (Naki et al., U.S. Pat. No. 5,798,358).
Apoptotic compositions with active agents selected from the group consisting of serotonin, caffeine, hydrocortizone, dexamethasone, dopamine, and gluquidone was proposed by Rubin et al. (U.S. Pat. No. 5,840,719). However, the presence of immunodepressive steroids can be detrimental in those patients who have chronic viral infections.
Development of an efficient cytokine blocker has also been explored but has experienced complications relating to the need to maintain their elevated level in many clinical situations.
All previous attempts in the prior art have concentrated on blocking or inhibiting apoptosis and lowering TNF-.alpha. levels or inhibiting its production. Methods of eliminating TNF-.alpha. were disclosed in U.S. Pat. No 5,605,690 to Jacobs et al. Inhibition of TNF-.alpha. production with concurrent increase in glucose levels was disclosed by Aston in U.S. Pat. No. 5,505,204.
Recent dinical and experimental data has provided compelling evidence of a much more complicated role of TNF-.alpha. in cell pathogenesis. In some ways these studies are completely contradictory to the conventional point of view that increased levels of TNF-.alpha. is harmful and leads to terminate stage of diseases.
Elevation of circulating TNF-.alpha. was protective against sepsis (Rigato O., Ujvari S., Castelo A., Salomao R., Infection; 24:314-318(1996)) and respiratory syncytial virus infection in vivo and in vitro (Neuzil KM., Tang YM., and Graham. Am. J. Med. Sci.; 311:201-204 (1996)). Even more surprising results were obtained after administration of TNF-.alpha., known to be a mediator of apoptosis and necrosis, was found to induce a protective effect against these conditions. Pretreatment with TNF-.alpha. significantly increased the tolerance to cerebral ischemia prior to ischemic episode. (Nawashiro H, et al., L. Cereb. Blood Flow Met.;17:483-490(1997)). This apoptosis cytokine, TNF-.alpha., inhibits anti-lgM-mediated apoptosis in Ramos cell. (Park E., et al., Exp. Cell Res.;226:1-10(1996)).
Based on the state of the prior art it can be seen that there is a considerably degree of controversy and uncertainty about the physiological role of TNF-.alpha.. All previous attempts to eliminate or inhibit TNF-.alpha. production do not reflect the precise pathogenic role of this cytokine, thus TNF-.alpha. elimination or level reduction carry potential dangers for patients due to obvious immunosuppression and undesirable antiapoptosis properties.
According to the present invention N-acetyl-D-glucosaminyI(.beta.-1-4)-N-Acetyl-muramyl-L-ananyl-D-isoglutami ne (GMDP) is used to modulate Fas mediated apoptosis and stimulate TNF-.alpha. production and selectively inhibit its p55(TNFR1).
GMDP was also isolated during analysis of the anti-tumor drug, blastolysine, which is a lysozyme cell wall hydrolysate of Lactobacillus Bulgaricus. (U.S. Pat. No. 4,395,399) GMDP has been extensively studied in animals, demonstrating adjuvant activity, antitumor activity, low pyrogenicity, and hypnogenic effect. (Andronova T., et al., Sov. Med. Rev. Immunol.;4:1-63(1991)).
GMDP and other muramyl dipeptides have shown inhibitory effects on lipopolysaccharide (LPS) induced TNF-.alpha., which results in preventing the toxic action of LPS during septic shock. (Adeleye T.A., et al., A.M.P.S.;102:145-152(1994)).
GMDP has been identified in human breast milk. it also has been suggested that GMDP enters the human body after the degradation of the probiotic part of the microflora and serves as a mediator of their systemic effect which could be clinically important. (Slesarev V., et al., PCT #97/05889). For example newborns fed with human milk are rarely infected by viral hepatitis. How and why the human milk prevents the viral hepatitis remains unknown.